从土壤中高效分离苏云金杆菌的方法

Method for Highly Efficient Isolation of Bacillus thuringiensis from Soil

在醋酸钠分离方法的基础上添加抗生素 ,能显著提高从土壤中分离苏云金杆菌的效率。当青霉素钠盐和硫酸庆大霉素的浓度分别达到 40 0︼g/ ml时 ,除地衣芽孢杆菌、短小芽孢杆菌和日本甲虫芽孢杆菌有微弱生长外 ,其它 9种杂菌均不能生长。对 2 9个 Bt亚种的生长抑制试验结果表明 ,仅 4个亚种有轻度至中度的生长抑制 ,但仍能形成孢晶。其它各亚种均能正常生长和形成孢晶。醋酸钠 -抗生素方法的可操作浓度为每克土壤稀释 2 0倍 ,较醋酸钠方法可操作浓度高 2 4倍。采用醋酸钠 -抗生素方法分离 Bt,总出菌率为 7.5 % ,高于醋酸钠方法总出菌率(3.8% ) ;分离菌株的基因类型较醋酸钠方法丰富 ,且相同基因型菌株的重复比例亦大大低于醋酸钠方法。

Based on sodium acetate method, the addition of antibiotics could significantly improve the isolation efficiency of B.thuringiensis from soil. B.licheniformis, B.pumilus, B.popilliae could grow slightly (colony was rather small), and other 9 contaminants could not grow when the concentration of sodium penicillin and gentamicin reached 400μg/ml. 4 subspecies out of 29 Bt standard strains were slightly or moderately inhibited, but still could form spores and crystals. The other subspecies could grow and form crystal normally. With this method, 10120 soil samples had been screened and 759 strains of B.thuringiensis had been isolated. The total isolation rate was 7.5%, which was higher than that of sodium acetate method (3.8%). Strains isolated by sodium acetate antibiotics method were primarily classified as 28 types based on gene combinations, in which 26.2% strains showed indeterminable gene types. The gene combination types of this method were richer than those of sodium acetate method. The strains with same gene types were also much lower than that of sodium acetate method. New genes might exist in the strains with indeterminable gene types which expressed 1 to several crystal proteins with novel molecular weight.